This proposal addresses the mechanical linkages within the cell that underlie cell migration and shape change. Such linkages are involved in invasion and metastasis by tumor cells; in regulated migration of leukocytes during immune surveillance and response; in human embryonic development; and in wound healing. Recent evidence indicates that cell motility involves a continuously acting cortical motor that engages and disengages, as with an automobile transmission with a clutch. Our goal is to understand the various mechanical couplings among the motor, the MT-rich bulk cytoplasm, and adhesions to the environment that underlie fibroblast motility during directed cell migration (Specific Aim 1), in non-migratory motility such as cell spreading (Specific Aim 2), and in cells poisoned with anti-cytoskeletal drugs (Specific Aim 3). These various motility states of fibroblasts are analyzed quantitatively for three sub-mechanisms of cell movement: cell adhesion, cortical motor activity, and engagement and disengagement of the motor to the cell surface and bulk cytoplasm. For example, we test the hypothesis that mechanical strength of adhesion is the result of recruitment of actin to the cytoplasmic side of the adhesion site, and that this actin recruitment, in turn, leads to engagement of the cortical motor to produce cytoplasmic movements. These analyses are carried out combining four methodologies: 1) calibrated glass needles to apply and measure forces to individual fibroblasts; 2) Visualization of the actin and microtuble cytoskeletons by transfection and expression of cytoskeletal proteins labeled cytoskeletal proteins labeled with green-fluorescent protein (GFP- technology); 3) Assessment of the continuously acting cortical motor through the centripetal movement of variously treated vinyl beads or calibrated needles adhering to the surface; and 4) manipulating the extent of cytoskeletal coupling to the motor and environmental by differential adhesion of surface-attached beads or needles. The proposed experiments are intended to substantially improve our understanding of the physical mechanisms of cellular motility and shape change, which understanding is poor compared to our knowledge of the molecules involved.